Purpose: :
Cigarette smoking (CS) is a powerful chemical oxidant and astrong epidemiologic risk factor for the development of Age-relatedMacular Degeneration (AMD). Previously, we showed that CS inducesphenotypic changes to the retinal pigmented epithelium (RPE)in a mouse model. Nrf2 is a transcriptional factor that controlsa cascade of antioxidant genes, and could protect the RPE fromCS induced oxidative insult. We hypothesize that Nrf2 will beactivated upon CS by RPE as apart of the cytoprotective response.The purpose of this project is to examine the Nrf2 cytoprotectiveresponse by the RPE after CS exposure.

Methods: :
ARPE-19 cells were exposed to cigarette smoke extract for 24hours. Nrf2 responsiveness was evaluated by nuclear translocationof Nrf2 protein by western analysis, Nrf2 response gene expressionby RT-qPCR, and complement activation by western analysis. Nrf2+/+and Nrf2-/- mice were exposed to CS for 1-21 days. The RPE/choroidwas extracted and analyzed by RT-qPCR for Nrf2 responsive geneexpression.

Results: :
Using the MTS viability assay, visually confluent ARPE-19 cellsexposed to 0-250mcg/ml CSE for up to 24 hours showed no evidenceof toxicity while 500-1000mcg/ml CSE caused a 20% and 80% reduction,respectively, in viability compared to DMSO control cultures.To demonstrate Nrf2 responsiveness in human RPE cells, Westernblot of cells exposed to 100 and 250 ng/ml CS Extract for 18hours showed a 2.25- and 2.50-fold increase, respectively, innuclear Nrf2 protein compared to vehicle treated cells (p<0.01for each dose of CS extract vs. vehicle). At this time point,a dose response effect of CS on Nrf2 responsive gene expression(NQO1, HO-1, and GCLM) was also observed (ANOVA, p<0.001compared to control, and 100 mcg/ml vs 250 mcg/ml; expressionwas normalized to β-actin.). CS can influence the complementcascade. Using similar culture conditions, while CSE 250mcg/mldid not induce C3 by Western analysis, CD46, an inhibitor ofC3, was reduced by 2-fold compared to untreated controls. Nrf2+/+mice (n=3 ) exposed for either 1 or 21 days of CS, had inductionof NQO-1 and GCLM in the RPE/choroid compared to mice raisedin air. There was no induction in Nrf2-/- mice raised in CSor air.

Conclusions: :
RPE cells in vitro and in vivo show evidenc e of Nrf2 nucleartranslocation and is associated with increased expression ofantioxidant genes. This acute response could be an importantprotective response by RPE cells to CS exposure.